Peripheral T-cell lymphoma, not otherwise specified is a heterogeneous group of aggressive neoplasms with indistinct borders. By gene expression profiling we previously reported unsupervised clusters ...of peripheral T-cell lymphomas, not otherwise specified correlating with CD30 expression. In this work we extended the analysis of peripheral T-cell lymphoma molecular profiles to prototypical CD30(+) peripheral T-cell lymphomas (anaplastic large cell lymphomas), and validated mRNA expression profiles at the protein level. Existing transcriptomic datasets from peripheral T-cell lymphomas, not otherwise specified and anaplastic large cell lymphomas were reanalyzed. Twenty-one markers were selected for immunohistochemical validation on 80 peripheral T-cell lymphoma samples (not otherwise specified, CD30(+) and CD30(-); anaplastic large cell lymphomas, ALK(+) and ALK(-)), and differences between subgroups were assessed. Clinical follow-up was recorded. Compared to CD30(-) tumors, CD30(+) peripheral T-cell lymphomas, not otherwise specified were significantly enriched in ALK(-) anaplastic large cell lymphoma-related genes. By immunohistochemistry, CD30(+) peripheral T-cell lymphomas, not otherwise specified differed significantly from CD30(-) samples down-regulated expression of T-cell receptor-associated proximal tyrosine kinases (Lck, Fyn, Itk) and of proteins involved in T-cell differentiation/activation (CD69, ICOS, CD52, NFATc2); upregulation of JunB and MUM1, while overlapping with anaplastic large cell lymphomas. CD30(-) peripheral T-cell lymphomas, not otherwise specified tended to have an inferior clinical outcome compared to the CD30(+) subgroups. In conclusion, we show molecular and phenotypic features common to CD30(+) peripheral T-cell lymphomas, and significant differences between CD30(-) and CD30(+) peripheral T-cell lymphomas, not otherwise specified, suggesting that CD30 expression might delineate two biologically distinct subgroups.
We report a case of an uveal melanoma patient with
p.Gly48Leu who responded to MEK inhibition. At the time of the molecular analysis, the pathogenicity of the mutation was unknown. A tridimensional ...structural analysis showed that Gα
can adopt active and inactive conformations that lead to substantial changes, involving three important switch regions. Our molecular modelling study predicted that
p.Gly48Leu introduces new favorable interactions in its active conformation, whereas little or no impact is expected in its inactive form. This strongly suggests that
p.Gly48Leu is a possible tumor-activating driver mutation, consequently triggering the MEK pathway. In addition, we also found an
p.Cys172Gly mutation, which was predicted by molecular modelling analysis to lead to a gain of function by impacting the Ig-like domain 2 folding, which is involved in FGF binding and increases the stability of the homodimer. Based on these analyses, the patient received the MEK inhibitor trametinib with a lasting clinical benefit. This work highlights the importance of molecular modelling for personalized oncology.
The bone marrow (BM) exists heterogeneously as hematopoietic/red or adipocytic/yellow marrow depending on skeletal location, age, and physiological condition. Mouse models and patients undergoing ...radio/chemotherapy or suffering acute BM failure endure rapid adipocytic conversion of the marrow microenvironment, the so-called "red-to-yellow" transition. Following hematopoietic recovery, such as upon BM transplantation, a "yellow-to-red" transition occurs and functional hematopoiesis is restored. Gold Standards to estimate BM cellular composition are pathologists' assessment of hematopoietic cellularity in hematoxylin and eosin (H&E) stained histological sections as well as volumetric measurements of marrow adiposity with contrast-enhanced micro-computerized tomography (CE-μCT) upon osmium-tetroxide lipid staining. Due to user-dependent variables, reproducibility in longitudinal studies is a challenge for both methods. Here we report the development of a semi-automated image analysis plug-in,
, which employs the open-source software QuPath, to systematically quantify multiple bone components in H&E sections in an unbiased manner.
discerns and quantifies the areas occupied by bone, adipocyte ghosts, hematopoietic cells, and the interstitial/microvascular compartment. A separate feature,
, fragments adipocyte ghosts in H&E-stained sections of extramedullary adipose tissue to render adipocyte area and size distribution. Quantification of BM hematopoietic cellularity with
lies within the range of scoring by four independent pathologists, while quantification of the total adipocyte area in whole bone sections compares with volumetric measurements. Employing our tool, we were able to develop a standardized map of BM hematopoietic cellularity and adiposity in mid-sections of murine C57BL/6 bones in homeostatic conditions, including quantification of the highly predictable red-to-yellow transitions in the proximal section of the caudal tail and in the proximal-to-distal tibia. Additionally, we present a comparative skeletal map induced by lethal irradiation, with longitudinal quantification of the "red-to-yellow-to-red" transition over 2 months in C57BL/6 femurs and tibiae. We find that, following BM transplantation, BM adiposity inversely correlates with kinetics of hematopoietic recovery and that a proximal to distal gradient is conserved. Analysis of
recovery through magnetic resonance imaging (MRI) reveals comparable kinetics. On human trephine biopsies
successfully recognizes the BM compartments, opening avenues for its application in experimental, or clinical contexts that require standardized human BM evaluation.
Background
Artificial genomic reference standards in a cytocentrifuge/cytospin format with well‐annotated genomic data are useful for validating next‐generation sequencing (NGS) on routine ...cytopreparations. Here, reference standards were optimized to be stained by different laboratories before DNA extraction and to contain a lower number of cells (2 × 105). This was done to better reflect the clinical challenge of working with insufficient cytological material.
Methods
A total of 17 worldwide laboratories analyzed customized reference standard slides (slides A‐D). Each laboratory applied its standard workflow. The sample slides were engineered to harbor epidermal growth factor receptor (EGFR) c.2235_2249del15 p.E746_A750delELREA, EGFR c.2369C>T p.T790M, Kirsten rat sarcoma viral oncogene homolog (KRAS) c.38G>A p.G13D, and B‐Raf proto‐oncogene, serine/threonine kinase (BRAF) c.1798_1799GT>AA p.V600K mutations at various allele frequencies (AFs).
Results
EGFR and KRAS mutation detection showed excellent interlaboratory reproducibility, especially on slides A and B (10% and 5% AFs). On slide C (1% AF), either the EGFR mutation or the KRAS mutation was undetected by 10 of the 17 laboratories (58.82%). A reassessment of the raw data in a second‐look analysis highlighted the mutations (n = 10) that had been missed in the first‐look analysis. BRAF c.1798_1799GT>AA p.V600K showed a lower concordance rate for mutation detection and AF quantification.
Conclusions
The data show that the detection of low‐abundance mutations is still clinically challenging and may require a visual inspection of sequencing reads to detect. Genomic reference standards in a cytocentrifuge/cytospin format are a valid tool for regular quality assessment of laboratories performing molecular studies on cytology with low‐AF mutations.
Cytological genomic reference standards are a valid educational and validation tool and show highly reproducible results. The detection of low‐abundance mutations is challenging and may require a visual inspection of sequencing reads.
Central nervous system involvement in Hodgkin lymphoma is extremely rare, especially in nodular lymphocyte predominant Hodgkin lymphoma (NLPHL), which usually carries a favorable prognosis. Here we ...report a case of a young patient with NLPHL, who developed a progressive and fatal neurological deterioration requiring a very extensive work-up including two biopsies to obtain the diagnosis of T-cell/histiocyte-rich large B-cell lymphoma like transformation. This report, which includes post-mortem analysis, highlights the correlations between clinical, radiological, and biological data but also the difficulties encountered in reaching the correct diagnosis.
We report postmortem cardio-pulmonary findings including detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in formalin-fixed paraffin embedded tissue in 12 patients with ...COVID-19. The 5 women and 7 men (median age: 73 years; range 35–96) died 6–38 days after onset of symptoms (median: 14.5 days). Eight patients received mechanical ventilation. Ten patients showed diffuse alveolar damage (DAD), 7 as exudative and 3 as proliferative/organizing DAD. One case presented as acute fibrinous and organizing pneumonia. Seven patients (58%) had acute bronchopneumonia, 1/7 without associated DAD and 1/7 with aspergillosis and necrotic bronchitis. Microthrombi were present in 5 patients, only in exudative DAD. Reverse transcriptase quantitative PCR detected high virus amounts in 6 patients (50%) with exudative DAD and symptom-duration ≤14 days, supported by immunohistochemistry and in-situ RNA hybridization (RNAscope). The 6 patients with low viral copy levels were symptomatic for ≥15 days, comprising all cases with organizing DAD, the patient without DAD and one exudative DAD. We show the high prevalence of DAD as a reaction pattern in COVID-19, the high number of overlying acute bronchopneumonia, and high-level pulmonary virus detection limited to patients who died ≤2 weeks after onset of symptoms, correlating with exudative phase of DAD.
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